Dissertation
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DISSERTATION Titel der Dissertation Community structure and distribution of functional microbial groups within two complex environments: microorganisms associated with marine sponges and potential sulfur-compounds-reducing microorganisms in peatlands. angestrebter akademischer Grad Doktorin der Naturwissenschaften (Dr. rer.nat.) Verfasserin / Verfasser: Doris Steger Matrikel-Nummer: 9803561 Dissertationsgebiet (lt. Stu- 444 Ökologie dienblatt): Betreuerin / Betreuer: Univ.-Prof. Dr. Michael Wagner Wien, am 18 März 2010 Formular Nr.: A.04 Contents Outline ......1 Part I Sulfate reducers in peatlands Introduction 5 Microorganisms with Novel Dissimilatory (Bi)Sulfite Reductase Genes are Widespread in Geographically Distant, Low-Sulfate Peatlands .21 Part II Hybridization regimes in DNA microarray experiments Introduction .....79 Spatial Gradients of Target Nucleic Acids Govern DNA Microarray Hybridization Assays ..87 Part III Microorganisms in marine Sponges Introduction 113 Sponge-Associated Microorganisms: Evolution, Ecology, and Biotechnological Potential .125 Diversity and Mode of Transmission of Ammoniaoxidizing Archaea in Marine Sponges .195 Part IV Summary ....205 Appendix ..211 Outline Part I of this thesis describes the analysis of dsrAB-carrying microorganisms in terrestrial wetlands. Newly developed tools such as a functional gene microarray, quantitative PCR assays and dsrB based denaturing gradient gel electrophoresis as well as clone libraries were applied to unravel the widespread occurrence of deep-branching dsrAB lineages, which outnumbered characterized dsrAB lineages in the Schlöppnerbrunnen fen system. Part II validates the signal variability in DNA microarray hybridizations. A simplified microarray format and numerical simulation tests were applied to investigate systematic variations in detected signal intensities and to explore underlying mechanisms of observed spatial gradients. Part III gives a comprehensive overview on the community structure of sponge associated microorganisms. A meta-analysis of all public available sponge derived 16S rRNA gene sequences as well as of new sequences retrieved from so far unstudied marine sponges was conducted. Moreover, the mode of transmission of Archaea probably involved in ammonia oxidation was analyzed by 16S rRNA and ammonia monooxygenase gene based clone libraries. The presence of detected putative ammonia-oxidizing archaea (AOA) in adult sponges and corresponding sponge larvae was confirmed by fluorescence in situ hybridization. Part IV includes a summary of the findings obtained in this thesis The Appendix includes a list of the author’s publications, oral and poster presentations, the acknowledgment, and the curriculum vitae. 1 Part I Sulfate reducers in peatlands Sulfate reducers in peatlands Introduction 1. Peatlands and their role in the global carbon cycle Wetlands are a common part of the global landscape, with mainly three distinguishing characteristics to other landform units: (i) the permanent or periodic presence of a water table at, near or above the land surface, which determines (ii) a unique soil development that is frequently characterized by low oxygen content and (iii) specialized plant communities that are adapted to these environments (Cowardin et al. 1995). Peatlands, also called organic wetlands, build up a substantial fraction of these systems and are probably the most widespread type of wetlands by occupying globally an area of about 4*106 km2 (Joosten et al. 2002). They represent ecosystems where net primary production exceeded the rate of decomposition and as a result predominately organic, carbon rich matter, so-called peat, accumulated over time. Their persistence depends on a constant long-term water input, and the origin of this water influences the form and function of peatlands. Based on their main water source, peatlands can be divided in predominantly groundwater-fed fens (minerotrophic peatlands) and in rainwater-fed bogs (ombrotrophic peatlands) (Bridgham et al. 1996). The source of water considerably influences the geochemistry of the peatland. Minerotrophic fens receive nutrient-input from incoming mineralized groundwater and thus higher concentrations of cations and anions are present compared to exclusively precipitation-fed bogs. Bogs are oligotrophic, as isolation from surrounding mineral soil results in low nutrient and mineral content, which is often associated with low pH values (< 5) (Bridgham et al. 1996). Dominating vegetation forms are Sphagnum mosses, sedges and ericaceous shrubs, which are adapted to these conditions (Vanbreemen 1995). Peatlands store about 15–30% of the world’s soil carbon (Turunen et al. 2002; Davidson et al. 2006). Thus, they are massive deposits of carbon, acting as a net carbon sink and contributing to global cooling in the past 8,000 to 11,000 years (Frolking et al. 2007). Undisturbed peatlands are likely to continue functioning as net carbon sinks at the decadal scale, despite the large interannual variability of individual peatlands (Moore et al. 1998). However, environmental and anthropogenic factors, such as climate change and land-use, affect the carbon balance of these ecosystems and can lead to substantial carbon loss (Dise 2009). Primary carbon inputs in 5 Part I peatlands derive from plant-assimilated carbon dioxide, whereby energy-rich organic compounds are deposited as litter or released below-ground by plant roots. Carbon loss is dominated by the emission of the gases carbon dioxide and methane (Keller et al. 2007). complex polymers exoenzymes denitrifiers - NO3 → N2 monomers manganese reducers Mn4+ → Mn2+ iron reducers redox potential primary fermenters Fe3+ → Fe2+ Acidiphilium-, Geobacter-, Clostridiaceae, Enterobacteriaceae, and Geothrix-related spp. Actinomycetales, sulfate reducers 2+ 2- SO4 → S Desulfomonile-, Desulforhopalus- and secondary fermenters acetogens Desulfosporosinus–related spp. Syntrophobacter-related spp. Clostridiaceae, Acidaminococcaceae methanogens CO2 → CH4 Methanobacterium-, Methanosaeta-, Methanosarcina- and Methanospirillum-related spp. Figure 1: Carbon mineralization processes in peatlands. Extracellular enzymes hydrolyze complex organic matter to monomers. Organic substrates, methanol or carbon dioxide are oxidized to acetate by acetogens or degraded by primary fermenters to organic acids, alcohols and amino acids. Resulting products are available either for secondary fermentation (syntrophic microorganisms) or for the microbial mediated sequential reduction of nitrate, manganese, iron (III), sulfate and finally carbon dioxide. Microorganisms identified in the Schlöppnerbrunnen fen are displayed. Soil microorganisms that use either oxygen or other electron acceptors as energy sources to decompose organic matter to carbon dioxide, methane and dissolved organic carbon play a major role in carbon effluxes from peatlands. As oxygen supply in peatlands is commonly restricted to small volumes, mainly anaerobic microbial mineralization using alternative electron acceptors takes place (Figure 1). Crucial factors influencing these decomposition processes are on one hand the availability of electron acceptors and on the other hand nutrient content and degradability of organic matter. In this context, the activity and potential environment-mediated inhibition of extracellular enzymes play a key role for substrate supply and microbial growth, as they hydrolyze complex organic matter to low molecular weight compounds (Makoi et al. 2008). Subsequently, acetogens are capable to oxidize a range of organic substrates (e.g. glucose), 6 Sulfate reducers in peatlands methanol or carbon dioxide with hydrogen as electron donor to produce acetate. Furthermore, primary fermenters degrade sugars to fermentation products, such as formate, lactate, butyrate and propionate. Resulting end products of these transformations are then available either for secondary fermentation catalyzed by syntrophic microorganisms or for several microbially mediated anaerobic reduction processes. Generally, a sequential reduction of nitrate, manganese, iron (III), sulfate and finally carbon dioxide is assumed, whereby respective respiration processes are regulated by differences in energy yields of the reactions (Achtnich et al. 1995). However, several studies have shown that respective processes can occur spatially and temporally at the same time in these heterogeneous and highly structured systems (Wieder et al. 1990; Yavitt et al. 1990; Koretsky et al. 2007). Processes using inorganic electron acceptors often do not fully explain total anaerobic mineralized carbon amounts in peatlands, and thus the presence of other organic electron acceptors, such as humic acids, and fermentation processes coupled to methanogenesis are supposed to contribute substantially to the decomposition of organic compounds (Lovley et al. 1996; Vile et al. 2003; Keller et al. 2007; Wüst et al. 2009). 2. Biogeochemical cycling of sulfur compounds in peatlands Microbial mediated transformations of sulfur compounds are closely linked to the carbon cycle as reducing processes are associated with organic matter utilization in anoxic habitats such as peatlands. Peatlands have water saturated anoxic and overlying oxic zones with fluctuating water tables, and thus dynamic sulfur cycling is expected (Figure 2). 7 Part I 2- O SO4 2 H2S DMS 2- atmospheric SO4 deposition su rf ac e inf low O2 2- 2- oxic R―SO4 SO4 H2S 2- SO4 assimilation Absorption on DOM minerals CBS 2- 2- S2O3 SO4 g rou ndw ate r anoxic flow 2- 2- SO4 H2S